Using a combination of second generation mRNA sequencing and bioinformatic approaches we have obtained a complete list of mRNAs expressed at each stage of development from the CFU-E stage to the enucleating erythroblast. We identified 14 genes that are strongly upregulated during this period and that encode transcription factors, chromatin-modifying enzymes, RNA Polymerase II elongation factors, or DNA binding proteins that have important roles in other developmental processes but whose functions in red cell development have never been explored: Runx1t1, Sertad2, SertadS, Mxd1, Mxd3, Btg2, Med13l, Ncoa7, Calcocol, Asf1b, Dedd2, Bag1, Hdac11, HEXIM1, and EII2. In Aim 1 we will determine which of these 14 proteins plays an important role in erythroid development from the CFU-E stage by systematically knocking down each in purified CFU-E cells and culturing them in the presence of Epo. Broad effects will be assayed by measuring proliferation, induction of CD-71 and Ter-119, nuclear condensation, enucleation, and accumulation of hemoglobin and other marker erythroid- important genes. In collaboration with the Zon laboratory we will knockdown each of these in zebra fish embryos and assess effects on erythropoiesis. In Aim 2, for HDAC2, Hipk-1 and -2, and the new factors that have the most dramatic effects on erythropoiesis when knocked down, we will determine the genes whose expression is directly and indirectly regulated by them, using second generation mRNA sequencing on cultured knockdown mouse progenitors. Finally, in Aim #3 we will determine the global roles of these factors on Polll binding to promoter regions, Polll elongation, and in some cases epigenetic histone modifications As example, using progenitors in which the factors have been knocked down, we will measure by Chip-seq the global distributions of Polll and two histone modifications characteristic of transcriptional elongation. Coupled with bioinformatic analysis we will determine whether control of erythroid- important gene transcription during erythropoiesis by each of these factors works at the level of Polll binding or Polll elongation. These and other studies will create an extensive framework for understanding the epigenetic and transcriptional regulatory networks active in terminal erythropoiesis.